Combination of atovaquone with proguanil for the treatment of protozoal infections

ABSTRACT

The invention relates to combinations of atovaquone and proguanil, their use in the treatment and propinylaxis of parasitic infections, such as protozoal parasitic infections, e.g. malaria and toxoplasmosis, and infections caused by P.carinii and their use in the manunfacture of medicaments for the treatment and/or prophylaxis of such infections. The combinations can conveniently be administered in a single pharmaceutical formulation. Preferably, atovaquone and proguanil are administered in a potentiating ratio so that they act in synergy.

This is a Rule 62 File Wrapper Continuation of application Ser. No.08/436,285, filed by May 15, 1995, now abandoned which is a 371 ofPCT/GB93/02425, filed Nov. 25, 1993

The present invention relates to synergistic combinations of2-[4-(4-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone(atovaquone) and proguanil which have anti-parasitic activity. Moreparticularly, the invention is concerned with pharmaceuticalcompositions containing said combinations, their use in the treatment ofprotozoal parasitic infections such as malaria and toxoplasmosis andtheir use in the treatment of infections caused by Pneumocystis carinii.

The compound2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone(atovaquone) has previously been disclosed, for example in EuropeanPatent No. 123,238 which relates to2-substituted-3-hydroxy-1,4-naphthoquinones of formula (I) ##STR1##wherein either R¹ is hydrogen and R² is selected from C₁₋₆ alkoxy,aralkoxy,C₁₋₆ alkyl-C₁₋₆ alkoxy, phenyl substituted by one or two groupselected from halogen and C₁₋₆ alkyl, halogen and perhalo-C₁₋₆ alkyl orR¹ and R² are both C₁₋₆ alkyl or phenyl, and n is zero or 1, andphysiologically acceptable salts thereof. The compounds are said to haveantiprotozoal activity. Specifically, compounds of formula (I) wherein nis zero are said to be active against the human malaria parasitePlasmodium falciparum and also against Eimeria species such as E.tenella and E. acervulina, which are causative organisms of coccidiosisand compounds of formula (I) where n is 1 are said to be active againstprotozoa of the genus Theileria, in particular T. annulata or T. parva.Amongst the compounds specifically named and exemplified is the compoundof formula (I) wherein n is zero, R¹ is hydrogen and R² is 4-chlorophenyl, i.e. atovaquone.

Proguanil is a well-known drug for prophylaxis, but not treatment, ofmalaria. It is one of the safest antimalarial drugs and may be given toyoung children and pregnant women. However, resistance of P. falciparumto proguanil has appeared, particularly in South East Asia, and is anincreasing problem.

In order to combat drug resistance, it is becoming standard practice touse combinations of more than one antimalarial, either simultaneously orsequentially. However, many such combinations are antagonistic,resulting in less effective treatment and the dosage regimens are oftencomplicated, increasing the likelihood of patients failing to completethe treatment. Accordingly, it was an object of the present invention toprovide a combination of antimalarial drugs which was not antagonisticand which did not require a complex dosing regimen.

It has now surprisingly been found that by combining, eitherconcomitantly or sequentially, atovaquone, represented in thisspecification by formula (II): ##STR2## and proguanil, potentiation ofantiparasitic and particularly antimalarial activity is achieved.Furthermore a potentiating combination of the compound of formula (II)and proguanil can be simply presented in a single pharmaceuticalformulation.

In a first aspect, the present invention provides a method for thetreatment and/or prophylaxis of a protozoal parasitic infection, e.g.malaria or toxoplasmosis, or an infection caused by P. Carinii inmammals, including humans, which comprises administering an effectiveamount of atovaquone or a physiologically acceptable salt thereof andconcomitantly or sequentially administering an effective amount ofproguanil.

In a second aspect, the present invention provides atovaquone for use inthe manufacture of a medicament, for administration either concomitantlyor sequentially with proguanil, for treatment and/or prophylaxis of aprotozoal parasitic infection, e.g. malaria or toxoplasmosis or aninfection caused by P. carinii, in mammals including humans.

Preferably the compound of formula (II) and proguanil are administeredconcomitantly. Most preferably the compound of formula (II) andproguanil are administered in a potentiating ratio.

Thus, according to a further aspect of the present invention, there isprovided a combination of atovaquone, or a physiologically acceptablesalt thereof, and proguanil wherein atovaquone, or its salt, andproguanil are present in a potentiating ratio.

The term `potentiating ratio` is used herein to indicate that atovaquoneand proguanil are present in a ratio such that the antiparasiticactivity of the combination is greater than that of either atovaquone orproguanil alone or of the additive activity that would be predicted forthe combination based on the activities of the individual components.Thus the individual components act synergistically in combinationprovided they are present in a potentiating ratio.

A potentiating ratio, which may be successfully used to treat malaria,including hydroxynaphthoquinone resistant strains of malaria is in therange 1:0.1-1:100 of proguanil:atovaquone. Suitably, the potentiatingratio is in the range 1:0.2-1:10.

A particularly preferred potentiating ratio is in the range 1:1-1:3.

The present invention also provides in another aspect a method for thetreatment and/or prophylaxis of malaria in mammals, including humans,which comprises administering an effective amount of a combination ofatovaquone, or a physiologically acceptable salt thereof, and proguanil.

The hydroxyl group of atovaquone may form salts with appropriate bases,and physiologically acceptable salts of atovaquone include inorganicbase salts such as alkali metal (e.g. sodium and potassium) salts andalkaline earth metal (e.g. calcium salts; organic base salts e.g.phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine anddiethanolamine salts: and amino acid salts e.g. lysine and arginine.

It will be appreciated that the compound of formula (II) may exist asthe cis or trans isomer, that is to say that the cyclohexyl ring may becis or trans substituted by the naphthoquinone nucleus and thechlorophenyl group. Both cis and trans isomers and mixtures thereof inany ratio may be used in accordance with the present invention. Ingeneral when the compound is in the form of a mixture of isomers thetrans isomer will be present in an amount of about 50% or will be thepredominant isomer but the use of mixtures in which the cis isomerpredominates is also included within the scope of the invention. Thespecific ratio of isomers may be varied as required; typical mixturesinclude those in which the cis/trans isomer ratio is about 1:1,40:60 and5:95. For use according to the present invention the trans isomer of thecompound of formula (II), or a mixture of its cis and trans isomerscontaining at least 95% e.g. 99% of the trans isomer, is preferred.

The compound of formula (II) may also exist in a tautomeric form inwhich the hydroxyl group donates its proton to one of the oxo groups andthe use of such tautomeric forms is included within the scope of thisinvention. However, it is believed that the stable form is that shown informula (II).

The amount of a combination of atovaquone and proguanil required to beeffective as an antiparasitic agent will, of course, vary and isultimately at the discretion of the medical or veterinary practitioner.The factors to be considered include the route of administration andnature of the formulation, the mammal's bodyweight, age and generalcondition and the nature and severity of the disease to be treated. Ingeneral, a suitable effective dose for administration to man fortreatment of malaria is in the range of 2.0 mg to 30 mg of proguanil perkilogram bodyweight per day and 0.5 mg to 30 mg of atovaquone perkilogram bodyweight per day, for example from 3 to 20 mg/kg/day ofproguanil and 1 to 20 mg/kg/day of atovaquone, particularly 5 to 15mg/kg/day of proguanil and 3 to 15 mg/kg/day of atovaquone.

A suitable effective dose for administration to man for prophylaxis ofmalaria is in the range of from 3 to 20 mg per kilogram bodyweight perweek of each of proguanil and atovaquone for example from 6 mg/kg/weekto 10 mg/kg/week of each of proguanil and atovaquone.

It should be understood that the dosages referred to above arecalculated in terms of the drugs per se.

For use according to the present invention the combination of atovaquoneand proguanil is preferably presented as a pharmaceutical formulation.

Pharmaceutical formulations comprise the active ingredients (that is,the combination of atovaquone and proguanil) together with one or morepharmaceutically acceptable carriers therefor and optionally othertherapeutic and/or prophylactic ingredients. The carrier(s) must beacceptable in the sense of being compatible with the other ingredientsof the formula and not deleterious to the recipient thereof.

Accordingly, the present invention provides a pharmaceutical formulationcomprising a combination of atovaquone and proguanil in association withone or more pharmaceutically acceptable carriers therefor.

The present invention further provides a process for the preparation ofa pharmaceutical formulation which process comprises bringing intoassociation a combination of atovaquone and proguanil with one or morepharmaceutically acceptable carriers therefor.

The combination of atovaquone and proguanil may conveniently bepresented as a pharmaceutical formulation in unit dosage form. Aconvenient unit dose formulation contains the active ingredients inamounts of from 10 mg to 3 g each, e.g. 50 mg to 3 g each. Typical unitdoses may contain for example 500 mg of atovaquone and 200 mg ofproguanil or 500 mg of atovaquone and 500 mg of proguanil.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal and parenteral(including subcutaneous, intradermal, intramuscular and intravenous),administration as well as administration by naso-gastric tube. Theformulation may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association the active ingredients with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of the active ingredients. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active compounds in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an inert liquid diluent. Tabletsmay be optionally coated and if uncoated, may optionally be scored.Capsules may be prepared by filling the active ingredients, either aloneor in admixture with one or more accessory ingredients, into the capsuleshells and then sealing them in the usual manner. Cachets are analogousto capsules wherein the active ingredients together with any accessoryingredient(s) are sealed in a rice paper envelope. The combination ofthe compound of formula (II) and proguanil may also be formulated asdispersible granules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged e.g.in a sachet. Formulations suitable for oral administration wherein thecarrier is a liquid may be presented as a solution or a suspension in anaqueous liquid or a non-aqueous liquid or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms e.g. tablets wherein the active ingredients are formulated in anappropriate release--controlling matrix, or are coated with a suitablerelease--controlling film. Such formulations may be particularlyconvenient for prophylactic use.

The active ingredients may also be formulated as a solution orsuspension suitable for administration via a naso-gastric tube.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of the active combination with thesoftened or melted carrier(s) followed by chilling and shaping inmoulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of the active combination inaqueous or oleaginous vehicles. Injectible preparations may be adaptedfor bolus injection or continuous infusion. Such preparations areconveniently presented in unit dose or multi-dose containers which aresealed after introduction of the formulation until required for use.Alternatively, the active ingredients may be in powder form which areconstituted with a suitable vehicle, such as sterile, pyrogen-freewater, before use.

The combination of atovaquone and proguanil may also be formulated as along-acting depot preparation, which may be administered byintramuscular injection or by implantation e.g. subcutaneously orintramuscularly. Depot preparations may include, for example, suitablepolymeric or hydrophobic materials, or ion-exchange resins. Suchlong-acting formulations are particularly convenient for prophylacticuse.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations for the various routes ofadministration described above may include, as appropriate one or moreadditional carrier ingredients such as diluents, buffers, flavouringagents, binders, surface active agents, thickeners, lubricants,preservatives (including anti-oxidants) and the like, and substancesincluded for the purpose of rendering the formulation isotonic with theblood of the intended recipient.

Compositions suitable for veterinary use include those adapted for oral,parenteral, and intrarumenal administration.

Methods for preparing atovaquone are described in EP 123,238, and onespecific method is illustrated in Example 1.

EXAMPLE 12-[trans-4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoguinone

a) 4-(4-Chlorolphenyl)cyclohexane-1-carbozxylic Acid

Acetyl chloride (30 g) and finely powdered aluminium chloride (60 g)were stirred together in carbon disulphide (120 ml) and then cooled to-50° C. in a CO₂ /oxitol bath. Cyclohexene (30 g), previously cooled to-50° C., was added dropwise during 10 minutes while maintaining thetemperature of the reaction mixture at below -20° C. The mixture wasstirred at -50° C. for a further 60 minutes and the solvent thendecanted to leave a gummy orange complex. A little chlorobenzene wasadded as the material warmed to ambient temperature; the remainder ofthe chlorobenzene (total 300 ml) was then added, the so-obtainedsolution heated at 40° C. for 3 hours with stirring, poured onto amixture of ice and concentrated hydrochloric acid and the organic layerseparated, washed with 2M hydrochloric acid, 2M sodium hydroxide andwater, dried over anhydrous sodium sulphate and evaporated to dryness.The product was distilled in vacuo, the fraction boiling at 140-154° C.(0.1 mm Hg) collected, diluted with an equal volume of petroleum ether(40-60), cooled to -6° C. and a continuous stream of nitrogen gasbubbled through, and the separated colourless solid recovered.

Bromine (2.8 ml) was added to a solution of sodium hydroxide (6.2 g) inwater (42 ml) at 0° C. The above-obtained substitutedhexahydroacetophenone (3.1 g) was dissolved in dioxan (15 ml) and thecold hypobromite solution then added, keeping the reaction mixture atbelow 20° C. The reaction mixture was stirred at ambient temperature for6 hours then allowed to stand overnight. Sodium metabisulphite was addedto destroy excess hypobromite, the mixture cooled and then acidified togive a colourless solid. The solid was filtered off, washed with water,dried and recrystallised from ethanol to give 4-(4-chlorophenyl)cyclohexane-1-carboxylic acid, m p. 254-256° C.

b) 2-[4-(4-chlorophenyl)cylohexyl]-3-chloro-1,4-naphthoguinone

A mixture of 2-chloro-1,4-naphthoquinone (3.95 g, 0.02 mol), 4-(4-chlorophenyl)cyclohexane-1-carboxylic acid (4.9 g, 0.02 mol) andpowdered silver nitrate (1.05 g, 0.0062 mol) was heated to reflux withvigorous stirring in 40 ml of acetonitrile. A solution of ammoniumpersulphate (12.0 g, 0.0525 mol) in 50 ml of water was added dropwiseover 1 hour. The mixture was refluxed for 3 hours then cooled in ice for30 mins, after which it was filtered, and the residual sticky solidextracted twice with boiling chloroform to remove inorganic material.The chloroform was removed by evaporation to leave a yellow-brown solid(ca 2.7 g). This was dissolved in 40 ml of boiling acetonitrile; alittle insoluble material was removed by filtration. On cooling, thetitle compound separated as yellow crystals, (550 mg) m.p. 172-175° C.

NMR, dH(δ₆ -DMSO) 8.05 (2H, mult., β-naphth), 7.85(2H, mult., a-naphth),7.30 (4H, s., PhH), 3.30 (1H, br.t., CH), 2.67 (1H, br.t., CH), 1.2-2.4(8H, mult., 4×CH₂).

c) 2-[4-(4-chlorochenyl)cyclohexyl]-3-hydroxy-1,4-naphthoguinone

The product of stage (b) was suspended in 10 ml of boiling methanol and0.55 g of potassium hydroxide in 5.5 ml of water was added dropwise over15 mins. The mixture was refluxed until a dark red solution formed.(after ca. 6 hrs) when 2 ml of concentrated hydrochloric acid wascautiously added dropwise. The mixture was cooled and filtered, and thesolid residue washed thoroughly with water. The water washings werere-acidified and filtered. The combined solid residues (500 mg) mp200-209°, were recrystallised from acetonitrile to give the titleproduct as the trans-isomer (300 mg) m.p. 216-519° C.

EXAMPLE 2

The following examples illustrate conventional pharmaceuticalformulations which may be employed in accordance with the presentinvention:

    ______________________________________                                        A.  Film-coated tablet                                                           Core:                                                                         Compound of Example 1 500 mg                                                  Proguanil hydrochloride 200 mg                                                Microcrystalline cellulose (Avicel PH101) 130 mg                              Hydroxypropyl cellulose, Lo-sub, (LHPC,LHll) 99 mg                            Sodium starch glycollate (Explotab) 30 mg                                     Povidone K30 36 mg                                                            Magnesium Stearate  5 mg                                                      Compression weight 1000 mg                                                    Coating:                                                                      Polymer dispersion 20 mg                                                      (Hydroxypropylmethylcellulose and titanium                                    dioxide and polyethylene glycol 400 and                                       colourant)                                                                    Polishing:                                                                    Polyethylene glycol 8000  2 mg                                                Total weight 1022 mg                                                       B.  Dispersible film-coated tablet                                               Core:                                                                         Compound of Example 1 500 mg                                                  Proguanil hydrochloride 200 mg                                                Microcrystalline cellulose (Avicel PH101) 100 mg                              Hydroxypropyl cellulose, Lo-sub, (LHPC,LHll) 83 mg                            Sodium starch glycollate (Explotab) 40 mg                                     Povidone K30 20 mg                                                            Magnesium stearate  5 mg                                                      Sodium docusate  1 mg                                                         Magnesium aluminium silicate (Veegum F) 50 mg                                 Sodium saccharin  1 mg                                                        Compression weight 1000 mg                                                    Coating:                                                                      Polymer dispersion 10 mg                                                      (Hydroxypropylmethylcellulose and titanium                                    dioxide and polyethylene glycol 400 and                                       colourant)                                                                    Polishing:                                                                    Polyethylene glycol 8000  2 mg                                                Total weight 1012 mg                                                       ______________________________________                                    

BIOLOGICAL TEST RESULTS EXAMPLE 3 Comparison of Drug Interactions inCombinations of Compound of Example 1 with Other Antimalarials.

In vitro drug sensitivity studies were carried out using thesemiautomated technique of Desjardins (Desjardins et. al. AntimalarialAgents and Chemotherapy 1979; 16(6):710-718). Antimalarial activity inthis system is assessed by inhibition of radiolabelled hypoxanthineincorporation into parasites by graded concentrations of drugs.

The antimalarial drugs for testing were dissolved in water, 95% ethanol,or DMSO; drugs dissolved in water were diluted 1:1 with 95% ethanol anddrugs dissolved in ethanol were diluted 1:1 with water. Drug solutionswere then diluted with culture medium containing 10% human serum tostarting concentrations 20-50 times the estimated IC₅₀ The drugs testedand their solvents are listed below:

    ______________________________________                                        Drug            Initial Solvent                                                                           Medium                                            ______________________________________                                        Compound of Example 1                                                                         DMSO        1640                                                Quinine Ethanol.sup.--  Water 1640                                            Chloroquine Water.sup.--  Ethanol 1640                                        Mefloquine Ethanol.sup.--  Water 1640                                         Primaquine Ethanol.sup.--  Water 1640                                         Artesunate Ethanol.sup.--  Water 1640                                         PM443 DMSO 1640                                                               Tetracycline DMSO 1640                                                        Norfloxacin DMSO 1640                                                         Ciprofloxacin DMSO 1640                                                       Proguanil Ethanol.sup.--  Water Lo-folate                                     Cycloguanil Ethanol.sup.--  Water Lo-folate                                   Pyrimethamine DMSO Lo-folate                                                  Trimethoprim DMSO Lo-folate                                                   Sulfamethoxazole DMSO Lo-folate                                               Dapsone DMSO Lo-folate                                                        Clopidol DMSO Lo-folate                                                       Allopurinol Ethanol.sup.--  Water Lo-folate                                   PS-15 DMSO Lo-folate                                                          WR99210 DMSO Lo-folate                                                      ______________________________________                                    

In order to study drug combinations. drug solutions at startingconcentrations were combined in various ratios (1:5,1:2.2:1 and 5:1).Drug solutions and combinations were then introduced into a 96-wellmicrotitre plate to give duplicate rows of compound of example 1, thedrug being combined and four combinations of the two drugs. Serial 1:3dilutions of the drugs with media were made to fill the 96-wellmicrotitre plate using a 12-channel pipetter. To evaluate drugsclassified as dihydrofolic acid reductase (DHFR) inhibitors, modifiedculture medium was used which contained only physiologic concentrationsof folic acid and PABA.

The remaining biological procedures were carried out according to theDesjardins technique except that three strains of P. faiciparum wereused (the multi-drug resistant W-2 clone, the drug-sensitive butmefloquine resistant D-6 clone and the C2B isolate resistant to thecompound of example 1) and incubation was extended for 72 hours.

Individual IC₅₀ s were calculated using the "MINSQ" program fromMicromath Scientific Software. Each set of paired data was fitted to thehyperbolic tangent function used by Desjardins.

The IC₅₀ s were normalised by assigning values of 1 to the IC₅₀ for thecompound of Example 1 and to the other drug being combined withproportional normalised values for each ratio of the two drugs beingstudied. An isobologram was constructed by fitting the data to theequation:

    Y.sub.i =1-[X.sub.i /(X.sub.i +e.sup.I *(1-X.sub.i))]

where Y_(i) =IC₅₀ for compound of Example 1 when combined with anotherdrug

X_(i) =IC₅₀ for another drug when combined with compound of Example 1

I=interaction parameter indicating degree of reversal

Values of I were calculated for each combination. Positive values of Iindicated a synergistic combination, negative values indicatedantagonism and I=O indicated additive interaction.

The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Drug combined                                                                   with compound of I                                                          Example 1   W-2           D-6     C2B                                         ______________________________________                                        Quinine     -1.36                                                               Chloroquine -1.84  -1.40                                                      Mefloquine -1.19                                                              Tetracycline 1.27 1.11 0.02, -0.08                                            Primaquine -0.79                                                              Artesunic acid  -0.18                                                         PM443  -1.28                                                                  Norfloxacin 1.02                                                              Ciprofloxacin -1.22                                                           Pyrimethamine 0.36  -0.48                                                     Trimethoprim 1.27  0.58                                                       Proguanil 2.43, 2.88 2.56 2.56                                                Cycloguanil 2.21 1.66 0.13, -0.73                                             Allopurinol 1.14 0.43                                                         PS-15 1.77, 0.65, 1.97 -0.74                                                  WR99210 0.02                                                                  Sulfamethoxazole 2.75                                                         Dapsone -0.39                                                                 Clopidol 2.38, 2.65 0.73                                                    ______________________________________                                    

The results show that combinations of2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone andproguanil exhibited most consistent potentiation compared to the otherdrug combinations tested. particularly against the hydroxynaphthoquinoneresistent C2B strain.

The optimum ratio of the combination with proguanil was estimated foreach of the three strains of malaria parasite by determining the ratioof the IC₅₀ of proguanil to the IC₅₀ of the compound of Formula (II).The results are given in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                        W-2     D-6     C2B                                           ______________________________________                                        Proguanil: Compound of Formula (I)                                                               920:1    4038:1  0.2:1                                        2473:1                                                                     ______________________________________                                    

EXAMPLE 4 Comparison of Anti-Toxoplasma Activities in vivo of Compoundof Example 1, Proguanil and Combinations Thereof

Activities of the compounds and combinations were examined in a mousemodel of T. gondii, using the increase in time to death and percentsurvival of the mice as measures of drug activity.

Groups of 10, 20 gm CBA/CA mice were infected orally by gavage with 6cysts of the C₅₆ strain of T. gondii, and drug treatment was started 3days later and continued for 10 days. All drugs were administered orallyby gavage. The following groups were examined:

Controls

Atovaquone @ 10 mg/kg

Atovaquone @ 25 mg/kg

Proguanil @ 25 mg/kg

Atovaquone @ 10 mg/kg+Proguanil @ 25 mg/kg

Atovaquone @ 25 mg/kg+Proguanil @ 25 mg/kg

All animals were examined twice dailv for 30 days and all deathsrecorded.

The results are shown in Tables 3 and 4:

                  TABLE 3                                                         ______________________________________                                        Mean Time to Death (days)                                                                  Atovaquone                                                                        0          10 mg/kg                                                                             25 mg/kg                                   ______________________________________                                        Proguanil   0    140        20.1   24.1                                         (mg/kg) 25 9.0 18.4 29.3                                                    ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        % Survival                                                                                 Atovaquone                                                                        0          10 mg/kg                                                                             25 mg/kg                                   ______________________________________                                        Proguanil   0    10.0       10.1   10.0                                         (mg/kg) 25  0.0  0.0 80.0                                                   ______________________________________                                    

The mean time to death of the control mice was 14 days, with only oneanimal surviving [10%]. (This animal may not have become infected withthe low inocculum used). Atovaquone alone increased this to 20.1 days at10 mg/kg and 24.1 days at 25 mg/kg, in both cases with a single survivor[10%]. Proguanil despite its excellent safety record in man, is toxic tomice. At 25 mg/kg, proguanil exhibits signs of toxicity, reducing themean time to death to 9 days, with no survivors. The combination of 10mg/kg atovaquone with 25 mg/mg proguanil gave a mean time to death of18.4 days. The combination of 25 mg/kg atovaquone and 25 mg/kg proguanilgave an increased time to death in spite of the toxicity of proguaniland 80% of the mice survived compared to the minimal survival of 10%mice given atovaquone alone.

EXAMPLE 5 Comparison of Anti-Toxoplasma Activities in vitro of Compoundof Example 1, Proguanil and Combinations Thereof

In vitro drug sensitivity studies were carried out using asemi-automated technique based on that used for malaria (Desjardins etal, Antimicrobial Agents and Chemotherapy 1979 16 (6) 710-718), bututilising the selective incorporation of 3[H]-uracil by T. gondii.Antitoxoplasma activity in this system is assessed by inhabition ofuptake of radiolabelled uracil into parasites by graded concentrationsof drugs.

The drugs were dissolved in DMSO and dilutions prepared using culturemedium containing 3% foetal calf serum. To study drug combinations, drugsolutions at starting concentrations were combined in various ratios1:1, 1:3, 3:1. Serial 1:2 dilutions of the drug solutions andcombinations were prepared and used in duplicate wells of a 96 wellplate previously seeded with HeLa cells and RH strain T. gondii. Drugswere added two hours after the parasite and the plates incubated at 37°C. for 24 hours when the 3[H]-uracil was added and incubation continuedfor a further 8 hours. The assay was completed by removing thesupernatant fluid, disrupting the T. gondii containing cells in SDS, andprecipitating the labelled proteins with TCA onto filter mats. Theincorporation of label was measured on a Beta plate scintillationcounter. Percent inhibition of uracil incorporation was calculated forthe compounds and combinations and IC₅₀ s calculated using the GS1programme. IC₅₀ s were normalised by assigning values of 1 to IC₅₀ forthe compound of example 1 and proguanil with normalised values for eachratio of the two drugs being studied. An isobologram was constructed byplotting these norrnalised IC₅₀ s against each other. Potentiation wasindicated by the values occuring below the line of the isobologram, anadditive effect by values on the line and antagonism by values above theline.

Plates were set up in triplicate and all values plotted.

The results are shown in table 5.

                  TABLE 5                                                         ______________________________________                                        Normalised IC.sub.50 s                                                          Atovaquone Proguanil Potentiation                                           ______________________________________                                        0.559278        0.583893 No                                                     0.196689 0.614094 Yes                                                         0.729381 0.253691 Yes                                                         0.345238 0.527273 Yes                                                         0.130952 0.6    Yes                                                           0.233333 0.118182 Yes                                                         0.404959 0.451538 Yes                                                         0.229201 0.768462 Yes                                                         0.244904 0.091538 Yes                                                       ______________________________________                                    

The results show that combinations of atovaquone and proguanil exhibitpotentiation in vitro against T. gondii.

EXAMPLE 6 Comparison of Anti-Pneumocystis Activities in vivo of Compoundof Example 1, Proguanil and Combinations Thereof

Activities of the compounds and combinations were examined in a saidmouse model of Pneumocystis pneumonia.

The level of infection of mice in each group was measured using standardlung impression smears and immunofluorescence tests. A score wasassigned to each mouse where 0=no infection and +4=very heavy infection.The results are shown in table 6.

                  TABLE 6                                                         ______________________________________                                                          No.      Mean                                                  Infected/ Score                                                              SCORE No. ± % of                                                         Treatment                                                                             0     +1    +3  +4  +4  Examined                                                                             SE      Control                        ______________________________________                                        Control 0     0     0   3   7   10/10  3.70 ± 0.14                                                                        100                              untreated                                                                     Atovaquone 0 0 5 5 0 10/10 2.50 ± 0.16 68                                  50 mg/kg                                                                      p.o. daily                                                                    Atovaquone 0 0 1 7 2 10/10 3.10 ± 0.17 84                                  25 mg/kg                                                                      p.o. daily                                                                    Proguanil 0 0 0 2 8 10/10 3.80 ± 0.13 85                                   25 mg/kg                                                                      p.o. daily                                                                    Atovaquone 5 4 1 0 0  5/10 0.50 ± 0.21 14                                  &                                                                             Proguanil                                                                     50 + 25                                                                       mg/kg p.o.                                                                    daily                                                                         Atovaquone 1 0 4 4 0 8/9 2.22 ± 0.31 60                                    &                                                                             Proguanil                                                                     25 + 25                                                                       mg/kg p.o.                                                                    daily                                                                       ______________________________________                                    

When dosed alone, atovaquone gave reductions in the infection score.Proguanil alone at 25 mg/kg/day was ineffective in prophylaxis of PCP inthe said mouse. Proguanil in combination with atovaquone showed synergy.

We claim:
 1. A method for the treatment of malaria in mammals whichcomprises administering2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone and1-(4-chlorophenyl)-5-isopropylbiguanide hydrochloride in the ratio of1:1 to 3:1.
 2. A method for the treatment of malaria in mammals whichcomprises administering2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone and 1-(4-chlorophenyl)-5-isopropylbiguanide hydrocloride in a ratio of 5:2.
 3. Amethod according to claim 1 or claim 2 wherein the2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone is in theform of the trans isomer or a mixture of cis and trans isomers in whichthe trans isomer predominates.
 4. A method according to claim 1 or claim2 wherein 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinoneis administered either concomitantly or sequentially with1-(4-chlorophenyl)-5-isopropylbiguanide hydrochloride.
 5. A methodaccording to claim 1 or claim 2 wherein the2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone and1-4(chlorophenyl)-5-isopropylbiguanide hydrochloride is administeredconcomitantly and in a single composition.